بسم هللا الرحمن الرحيم

بسم هللا الرحمن الرحيم -Please refer to the slides from (4-20) -Slides (4, 5) -Oxidative phosphorylation consists of 2 parts: 1.electron transport chain (series of electron transport proteins much filled up with hydrogen gradient known as proton motive force which forms energy across the membrane, so proton will move according to their chemical gradient, but they can t pass through the membrane, they pass through a protein molecule called ATP synthase in order to synthesis Atp molecules). 2. ATP synthase 1 P a g e

-Slides (6, 7, 8) - 3 types of ET (electron transport) occur in OxPhos: 1. Direct ET, as in the reduction of Fe+3 to Fe+2 2. Transfer as a hydrogen atom {(H+) + (e-)} 3. Transfer as a hydride ion (: H-) -Electron transport molecules: -In order to transport electrons from one molecule to another (from one protein to another ),there must be something transport them which are electron transport molecules ( electron carrying molecules ),e.g.: nicotinamide molecules (NAD, NADP ), flavins (FMN, FAD ). -electron carrying molecules which related to the electron transport chain: 1. NADP, NAD: they are free in the solution (they can move freely), water-soluble -NADH: carries electrons to NADH dehydrogenase -NADPH: generally supplies electrons to anabolic reactions -Neither can cross the IMM (inner mitochondrial membrane) 2. Flavoproteins (FMN (flavin mono nucleotides), FAD): they are tightly bound to the proteins. -Slides 9 3. Ubiquinone: Also called coenzyme Q, has a cyclic diene(cycle contain 2 double bonds) structure carrying 2 oxygen molecules on the ring, so it can accept 2 electrons in the form of 2 H+( 2e + 2H+ ), finally it takes 2 hydrogen molecule, so the oxygen on the ring become OH. -Can accept either 1 e- or 2 e- 2 P a g e

- Lipid-soluble benzoquinone with a long isoprenoid side chain, so it can transport electrons through the IMM (because it is lipid-soluble) -semiquinone radical: the electron make a free radical, ubiquinone which is partially reduced is hazardous because it has free non-bound electron which is a free radical. infraction) patients Sometimes prescribed for recovering MI (myocardial -Slide (10, 11) 4.cytochromes : proteins that contain hemes ( hemes are different types, different from each other in structure,their structure make them different under spectrometry (spectrometry : put protein inside solution and put it under a device which is spectrometry, and every heme has a specific reflection differ from the others ), Classification of hemes based on light absorption. - Hemes has a characteristic reflection gives you band within the visible light around 400 nm, so all proteins contain heme are in red color. -Mitochondria contain three classes o f cytochromes (a, b, & c) - (Heme a) and (heme b) are non-covalently bounded to the protein by affinity,( heme c) is covalently bounded to the protein. - Cytochromes are named according to the heme they contain. -any heme can go under 2 stages (oxidized, reduced), any heme contain iron (iron when oxidized +3, when reduced +2), so any heme protein can go reductionoxidation reaction. - Heme which oxidized give us one peak around 400 nm, if it reduced it gives us 3 peaks (alpha, beta, gamma). 3 P a g e

-hemes are divided to (a,b,c ) according to wavelength of alpha band,( heme a ) give you alpha band around 600 nm, (heme b) give you alpha band around 560 nm, (heme c) around 550 nm. -when they come to name hemes, they don t found anymore names, so they named it by the exact wavelength band (Cytochrome b562; Cytochrome c550; Cytochrome c551). -heme as a missionary (transporter) can carry one electron because it can make a reduction to (iron +3) to (iron +2). - ΔE: redox potential (the capability of losing or accept electron) isn t defined for the hemes, it s different from one heme to another depending on the micro environment present around the heme. - hemes present in complex 3(cytochrome bc complex),which are ( heme b and c ) are transmembrane (spanning the whole membrane at both sides ), complex 4 ( heme a and heme a3 ) which are transmembrane, so heme( a,b,c ) in cytochromes are spanning the membrane except free cytochrome c which present on outer surface of mitochondrial membrane. 4 P a g e

Slide 12 5. iron-sulfur complexes (Fe-s complexes): molecules has the ability to carry electron and donate electrons, because it has an iron molecule, the complex has an specific arrangement from the sulfur and iron making a specific shape. -Types of iron-sulfur complexes: 1. One iron with 4 cysteine residue (4 sulfur atoms) 2. 2 irons and 2 inorganic sulfur and 2 cysteine, so each iron is connected to 4 sulfur atoms 3. 3 iron with 4 sulfur (each iron atom connected with 4 sulfur atoms) 4. 4 iron with 4 sulfur atoms. -Modification could happen on iron-sulfur complexes, like replacing histidine with cysteine (rieske iron- sulfur proteins), (2 His instead of 2 Cys) -Iron-sulfur complexes have the ability to transfer one electron at a time. -at least, we have 8 iron-sulfur complexes in electron transport chain, 7 present in complex 1,and the other in complex2,and others. -redox potential of iron-sulfur complexes are the same as hemes and as flavins, it isn t defined,depend on the micro environment around the protein, it ranges From(-65 to 450 mili volts),wide range due to micro environment. 5 P a g e

-slide 13 -oxidative phosphorylation consists of 2 parts: 1. Oxidative part: redox reactions (ETC) 2. Phosphorylation part: phosphorylation of ADP to make ATP (ATP synthase) -so in order to have oxidative phosphorylation we need: electron carrying molecules, electron donor molecules (NADH, FADH2 which coming from Krebs cycle), electron acceptor molecule (final acceptor molecule is oxygen). - Requirements for oxidative phosphorylation in order to happen: 1. intact inner mitochondrial membrane: because that the purpose of the pathway of electron is to produce energy, we use the energy of electron to pump protons outside the inner membrane in order to make proton electrochemical gradient, so intact means that inner mitochondrial membrane must be impermeable to proton in order to keep a higher concentration of proton outside the inner membrane of mitochondria. 2. ATP synthase: which is a machinery protein that transfers the energy in the electrochemical gradient of protons into energy to phosphorylate (ADP to ATP)? (Electron transport chain + ATP synthase = oxidation phosphorylation) Slide 14 -Chemiosmotic theory : theory was put to explain ATP synthesis that result at the end from the transfer of electrons.(with every movement of electron in this 6 P a g e

pathway,they lose energy almost from one complex to another around 16 kcal/mol, this energy used to pump protons outside the inner membrane, making electrochemical gradient (proton motive force ). -proton motive force pumped in significant amount from 3 sites, when they pass back, they pass through pores because the membrane isn t preamble to protons, and this pores represented by special complexes we called it ATP synthase which is complex 5. The complexes that transport electrons: -Slide 15 COMPLEX 1: it s called NADH dehydrogenase, why? Because it dehydrogenases the NADH, & it s called (NADH- quinone oxidoreductase) why? Because it oxidizes the NADH & reduce quinone. -So the protein NADH dehydrogenase is the first complex which bind NADH, this protein is a large complex which have at least 25 polypeptide chain (25 protein subunits gathered together in a huge complex, may reach to 42 in number ), it is a huge flavoprotien, it is membrane spanning molecule, why its called flavoprotien? Because it contains flavin mononucleotide (FMN) inside it, so Complex 1 is a huge flavoprotein because it contains an FMN which is tightly bounded protein. -Note: Also succinate dehydrogenase (complex 2) contains flavoprotein (FAD). -At least it contains 7 iron- sulfur complexes which used for passing electrons from 1 molecule to another to pass them from (NADH) finally to (the quinone) - First of all, electrons will be donated from NADH to FMN, FMN after accept the electrons become FMNH2, then it passes the electrons to iron- sulfur complexes then to the quinone. 7 P a g e

Note: when e- pass from NADH to quinone there is a drop of energy almost (13 to 14 Kcal), this energy used to pump 4 protons out of complex 1 for 2 e- passing through it. Slide 16 COMPLEX 2: called (succinate dehydrogenase) & it s part of Krebs cycle. -Note: Complex (1, 3, 4) spanning the inner mitochondrial membrane but complex 2,not spanning the inner membrane but attached to the inner mitochondrial membrane. Note: the only physical binding mean that connecting Krebs cycle with ETC is complex 2. -Important: NADH & NADPH is freely movable e- transport carriers, they are water soluble, can be dissolved in any solution (can move in the solution by themselves) but FAD is not like this, FAD is always binding to the protein so it present inside complex2, its part of complex 2. -In complex 1 we can say that e-transport from NADH to complex 1 because NADH is free movable come to the inner membrane give e- transforming to NAD+, but in complex2 we can t say that because FAD is part of complex 2, so FADH2 not donating e- to complex 2, it donates it to iron- sulfur complex and finally to the quinone. 8 P a g e

-( Complex 1) and (complex2) gives e- to ubiquinone result in reduced ubiquinone and this ubiquinone pass e-to (complex 3). -Note: not only succinate dehydrogenase can donate e- to the quinone, also there is a flavoprotein that contain flavin (FAD) can give e-to coenzyme Q(quinone) & it has the capacity to transfer e- to ubiquinone directly ( this protein that contain FAD present in the inner membrane) -Q: How much excess energy can be given from e- passing from complex 2 to quinone? zero Kcal ( this is the exception because their potential energy closely similar to each other, so it will not be capable of pumping protons), so in complex (1,3,4) we have pumping of protons but in (complex 2)we don t have proton pumping machinery because 1- we don t have an excess energy 2- the other reason that complex (1,3,4) are spanning proteins (spanning the whole membrane) so they have a pathway for transferring protons, even if (complex 2) have an excess energy its attached to the inner surface,not spanning membrane, so doesn t have a pathway to transfer protons. Note: with each transfer of e- it give (13 to 14 Kcal) energy except in complex 2. -Slide (17, 18) (here you must refer to the pictures in the slides in order to understand) COMPLEX 3: is a dimer (2 monomers gathered together) (complex 3) called cytochrome bc1 complex because it contains 2 types of heme (b,c) & also called Q-cytochrome c oxidoreductase because it oxidizes the quinone & reduces cytochrome c, so it catalyzes the transform of e- from the reduced quinone to the cytochrome c ( complex 3 take e- present in the quinone & give them to cytochrome c ) -it contains cytochrome b (that contain 2 hemes b inside it 1.heme bl & heme bh) & contain cytochrome c (that contain heme c1) 9 P a g e

-(complex 3) composed of 11 subunit, including : (cytochrome b) and ( cytochrome C )and contain at least (1 iron- sulfur center),and as we say before 2 cytochromes contain 3 heme groups ( 2 hemes present in cytochrome b (bl,bh) & 1 heme present in cytochrome c (c1)) -it contains 2 quinone binding sites & 1 cytochrome c binding site. -Per 2 e- moving through (complex 3), four protons passing outside as in complex1 -NOTE: In complex 1 for 2 e- passing, 4 protons pumped In complex 2 for 2 e- passing, pump nothing (zero) In complex 3 for 2 e- passing, 4 protons pumped In complex 4 for 2 e- passing, 2 protons pumped - NOTE: So for every 2 e- moving through ETC there is pumping for 10 protons -The process that happen inside complex 3: 2 e- come to (complex 3) from ubiquinone( ubiquinone has the ability to transfer 2 e- per time),then complex 3 donate 1 e- to the cytochrome c because cytochrome c contain heme & heme capable to transfer 1 e-. Q: Dr asked again from where e- comes to complex 3 & where it donates them? e- Come from ubiquinone (quinone carry 2e- where as heme carry 1e-) Q : how you can explain what happened to the second e- after this pathway (( 2e- pass from NADH to complex 1, then this 2e- to quinone then to (complex 3), then (complex 3) give 1e- to cytochrome c)), where the other e- go??? First we all know that free e- are bad & hazardous inside the human body because they 10 P a g e

considered free radicals ( which mean reactive oxidative species which mean cancer ) -Dr focused again & again on the issue of how to explain why for each 2eenter the ETC and 1e- exit & why for 2e- enter 4 protons exit, to explain this we have to know quinone cycle, so reduced ubiquinone come with 2e- in the form of 2H(QH2) to complex 3 ( 2e- enter complex3, iron- sulfur complex can transport 1eand heme can transport 1e- ). - So 1. First e- from the quinone bind to iron- sulfur complex to heme c1 to cytochrome c to complex 4 and 2. the second e- where it go, do we have to wait until the cytochrome c return from complex 4 after giving it the first e-, but that doesn t happen in the body, second e- come to (heme bl and heme bh) in cytochrome b (first to heme bl then to heme bh finally to oxidized quinone molecule producing semiquinone ) -1e- go to cytochrome c & the other e- go to free quinone molecule producing semiquinone but this molecule is hazardous, how to solve this problem??? there is another reduced quinone come from NADH carrying 2e- goes on the same pathway to complex3, 1e- of them go to iron- sulfur complex, then to heme c1 then to cytochrome c & the other e- go to the semiquinone turning it to quinone (a new QH2, the semiquinone when it take 1e-it take also 2 protons (2H+) becoming QH2 & that s why complex 3 pumps 2H+ outside). These 2 notes are very important: 1- SO in the result: 2 reduced quinone molecules with 4e- from NADH enter and one reduced quinone regenerates (that means 2 molecules enter and 1 molecule out so the net result that we used 1 molecule only) 2-SO per 4e- enter, I used 2e- and donate them to cytochrome c (per 2e- moving, I made 2 cytochrome c)and the other 2 e- used to regenerate reduced quinone, so we conclude that we use 2 e- to move 2 cytochrome c. 11 P a g e 2 ubiquinone enter, 1 ubiquinone regenerated 4e- enter, 2e- regenerated

-NOTE: fully reduced quinone with 2e-, fully oxidized quinone with zero e-, semiquinone with 1e-. -Net equation: QH2 + 2 Cyt c1 (oxidized) + 2H+ (result in) Q + 2 cyt c1 (reduced) + 4H+ -So Q-cycle accommodate the switch between 2e-/1e- and explain the measured 4H+/2e- so it means that I repeat the cycle 2 times & by this repeating I regenerate one of the reactant so I use the reactant for one time as a net result. Slide (19, 20) COMPLEX 4: called (cytochrome c oxidase) because cyt c passes e- from (complex 3) to (complex4), complex 4 which take e- from cyt c oxidizes it; it passes e- from cyt c to O2 -Complex4 contains 2 copper sites as e- transporting molecules & contains 2 hemes (heme A & heme A3) & contains oxygen binding site. -O2 finally reduced to become 2H2O ( O2 give 2H2O ) ( to reduce O2 to 2H2O we need 4e-, 4e- means 4 cyt Cs & need 2 NADH to reduce water ) -For theoretical reasons, per 2e- moving I m reducing half O2 to H2O Cyt c (reduced) + 4H+ + O2 (result in) cyt c (oxidized) + 2H2O -Cytochrome c oxidase (complex 4) has a much lower Km for O2 than myoglobin (dr asked what the mean of Km??? Km determine the affinity, which mean that if a molecule has a lower Km it can reach 50% of saturation with lower concentration, so it has higher affinity) -So Km of (cytochrome c oxidase) must be lower than myoglobin so it can take O2 from myoglobin to ETC. 12 P a g e

- -the process that happen inside complex4: now we have 2 coppers ( A,B) & 2 hemes ( cyt.a & cyt.a3 ), ( copper A faraway from heme A so work separately & cyt. A3 is close to copper B so work as one unit), the first e- bind to (copper A) reducing it from (Cu+2 to Cu+1) then from copper A to cyt. A then to the complex (heme b with cyt A3), other e- come but copper already reduced, so where it go?? to the heme & reduced it ( reduced heme can bind O2 with high affinity, but oxidized heme bind oxygen with low affinity ), now iron close to copper so iron that carry oxygen come and bind to another oxygen,then copper make bridge with oxygen so we have (iron-o-o- Cu), the third e- used to cleavage of O-O bond result in oxygen bound to iron & oxygen bound to copper, the fourth e- convert (O-2) to OH- then add 2H+ producing 2H2O. Done by: Mustafa Abu Al-kishik 13 P a g e

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